CN103215254A - Method for extracting activated sludge sample DNA (Deoxyribonucleic Acid) in SBR (Styrene Butadiene Rubber) - Google Patents

Method for extracting activated sludge sample DNA (Deoxyribonucleic Acid) in SBR (Styrene Butadiene Rubber) Download PDF

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CN103215254A
CN103215254A CN2013101513518A CN201310151351A CN103215254A CN 103215254 A CN103215254 A CN 103215254A CN 2013101513518 A CN2013101513518 A CN 2013101513518A CN 201310151351 A CN201310151351 A CN 201310151351A CN 103215254 A CN103215254 A CN 103215254A
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dna
sample
active sludge
sbr
described step
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赵晓祥
张湾
邓航
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Donghua University
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Donghua University
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Abstract

The invention relates to a method for extracting activated sludge sample DNA (Deoxyribonucleic Acid) in SBR (Styrene Butadiene Rubber). The method comprises the following steps of: sludge sample pretreatment, crushing, removal of protein, separation and purification of DNA, purification of genome DNA, ultraviolet spectrophotometric analysis of DNA and agarose gel electrophoresis detection of DNA. The method for extracting activated sludge sample DNA in SBR is quick and efficient, and capable of extracting activated sludge genome DNA in dyeing and printing water or waste water under the same water quality condition efficiently; and the method has good application prospect.

Description

The extracting method of active sludge sample DNA among a kind of SBR
Technical field
The invention belongs to the extraction field of DNA, the extracting method of active sludge sample DNA among particularly a kind of SBR.
Background technology
The DNA purity difference that the different methods that bacteria total DNA is extracted in the active sludge obtains, the purity of analyzing DNA is used to understand the structure of community of microorganism, to identify and separation process system in dominant bacteria very important.Make every effort to improve biological treatment dye wastewater treatment ability by this many-sided research.DNA extraction is the precondition of molecules technology.Only extracted enough and DNA that purity suits, just can carry out follow-up experiment and research, so the extraction of DNA can be described as molecular biological key point.
About bacteria total DNA extracting method in the active sludge: when extracting activated sludge DNA, it is very essential that sample is carried out pre-treatment.Contain impurity such as a large amount of humic acid, fulvic acid and heavy metal ion in the active sludge, also has the outer free DNA of some born of the same parents, they all greatly influence DNA extraction and follow-up work, adopt TENP and PBS damping fluid that mud sample is carried out pre-treatment, can remove impurity such as the humic acid in the mud sample, various ion, inorganics and organism, reduce the pollution of the outer dissociative DNA of born of the same parents.
Lysis is the process of destroying cell walls and cytolemma.Cleavage method commonly used has chemistry, enzyme and mechanical lysis.This experiment adopts the chemical cracking method directly to extract genomic dna from wastewater sample.What proteic removal was used is traditional phenol chloroform extraction method.The precipitation of nucleic acid is typically chosen in ethanol, the Virahol equal solvent carries out, and it is lower to use Virahol can obtain the content of the DNA of high yield and humic acid, so select for use Virahol to come deposit D NA in this experiment.The DNA that precipitation obtains can contain impurity such as humic acid and phenolic compound usually, and these impurity can have influence on follow-up analysis, particularly pcr amplification.The humic acid of low amount can suppress the PCR reaction, makes it can not get spawn.
Summary of the invention
Technical problem to be solved by this invention provides the extracting method of active sludge sample DNA among a kind of SBR, this method has fast, characteristics of high efficiency can the high efficiency extraction dyeing waste water or the active sludge genomic dna of identical water quality situation waste water, has a good application prospect.
The extracting method of active sludge sample DNA among a kind of SBR of the present invention comprises:
(1) all product of active sludge mixing at first before processing is evenly distributed suspended matter wherein; Adopt TENP, PBS solution and granulated glass sphere concussion method that sample is carried out pre-treatment; To adopt the freeze-thaw method fragmentation through pretreated mud sample, refrigeration, boiling water boil, and the SDS damping fluid that adds mass percent concentration 2%～10% is again removed protein;
(2) Virahol of 0.6 times of sample volume of adding is put upside down mixing, precipitates; Centrifugal, outwell supernatant liquor; Natural air drying, with TE damping fluid suspended sample, dissolution precipitation promptly gets the genomic dna crude extract; Add RNase A, 35～40 ℃ of temperature are bathed 15-20min digestion RNA, adopt DNA glue to reclaim test kit then, carry out purifying according to operation instructions;
(3) measure the absorption photometric value of above-mentioned purify DNA sample with ultraviolet spectrophotometer at wavelength 230,260,280nm place; Above-mentioned purify DNA sample is carried out agarose gel electrophoresis, behind the ethidium bromide staining, take pictures with the ultraviolet gel imaging system.
TENP liquor capacity in the described step (1) and PBS liquor capacity are 3～5 times of sewage sample; The granulated glass sphere diameter is 1～2mm, the centrifugal 10～30min of 3000～5000rpm.
Refrigeration, boiling water in the described step (1) boil and is specially: place-25～-15 ℃ of refrigerator and cooled to hide 1～2h, boiling water boils 10～20min, repeats once.
After refrigeration, boiling water boil in the described step (1), add 2～4ml Extraction buffer, 1～5 granulated glass sphere, whirlpool 5～10min.
The mass percent concentration of the SDS damping fluid in the described step (1) is 2%.
Precipitation in the described step (2) is specially puts into the refrigerator precipitation 1～2h or the precipitation of spending the night.
The consumption of the TE damping fluid in the described step (2) is 100 μ L.
The concentration of RNase A in the described step (2) is 10mg/mL, and consumption is 4 μ L.
Sepharose mass percent concentration in the described step (3) is 1%, and voltage is 100～110V, electrophoresis 0.5～1h.
Employing adds the 2%SDS damping fluid and adds the 10%SDS damping fluid and contrast, and in conjunction with freeze-thaw method the sample after the pretreatment process processing is analyzed comparison according to top result, and the extraction effect that adds 2%SDS is better than 10%SDS.
The present invention is a research object with dyeing waste water SBR system, to the active sludge sample that in reactor, extracts through or without the mud sample under two kinds of situations of pre-treatment, adopt granulated glass sphere concussion method and two kinds of lysis methods of freeze-thaw method, the influence of DNA extraction effect more separately, in the hope of set up a kind of fast, efficient and be applicable to the active sludge extracting genome DNA novel method of molecular biology subsequent analysis.The result who detects by absorbancy and agarose gel electrophoresis shows: active sludge adopts freeze-thaw method to add the DNA extraction operation of 2%SDS lysing cell after TENP and PBS pre-treatment, can obtain the activated sludge DNA sample that quantity is many, purity is high.
Beneficial effect
The present invention has fast, and characteristics of high efficiency can the high efficiency extraction dyeing waste water or the active sludge genomic dna of identical water quality situation waste water, has a good application prospect.
Description of drawings
Fig. 1 is the DNA electrophorogram that embodiment 1 Different Extraction Method obtains; Wherein, Marker: λ DNA-Hind III; 1: pre-treatment+granulated glass sphere concussion; 2: pre-treatment+freeze thawing; 3: be untreated+the granulated glass sphere concussion; 4: be untreated+freeze thawing;
Fig. 2 is the DNA electrophorogram that embodiment 1 different lysis methods are extracted; Wherein, Marker: λ DNA-Hind III; 5,6: freeze-thaw method+2%SDS damping fluid; 7,8: freeze-thaw method+10%SDS damping fluid;
Fig. 3 is embodiment 1 active sludge total DNA extraction result; Wherein, 1,2,3 to be respectively the anoxic section time be phase sample before, during and after the 2h; 4,5,6 to be respectively the anoxic section time be phase sample before, during and after the 3h; 7,8,9 to be respectively the anoxic section time be phase sample before, during and after the 4h; Marker: λ DNA-Hind III.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Embodiment 1
One, the source of active sludge sample and pretreatment process
1, source
Early stage, mid-term, the later stage of different anoxic time periods of SBR system take a sample, and the active sludge sample is taken from the reaction tank muddy water mixed solution.
2, pretreatment process
Sample is at first wanted mixing before processing, suspended matter wherein is evenly distributed.Get about 10mL sewage sample, add the sterilization centrifuge tube, 4000rpm, centrifugal 20min, collecting precipitation.Add about 4 times of volumes TENP buffer (50mMTirs, 20mM EDTA, 100mM NaCl, 0.01g/mL PVP pH10.0), adds fully homogenizing (separating flocculation) of granulated glass sphere (d:1mm).4000rpm, centrifugal 20min abandon supernatant (repeating twice, until clarification of supernatant liquor).The PBS buffer (8g NaCl, 0.2gKCl, the 1.44gNa that add 4 times of volumes 2HPO 4, 0.24gKH 2PO 4, be dissolved in 1L water, pH7.4), whirlpool mixing, 4000rpm, centrifugal 20min, collecting precipitation (repeating twice).
Two, freeze-thaw method cracking
To place-20 ℃ of refrigerator and cooled to hide 1h through pretreated mud sample, boiling water boils 10min, puts into-20 ℃ of refrigerator and cooled again and hides 10min, and boiling water boils 10min.The centrifugal 20min of bacterium liquid 4000rpm that will obtain through above-mentioned lysis method abandons supernatant liquor, and throw out is used for further cracking.In throw out, add 2ml Extraction buffer(100mM EDNA, 100mMTirs-base, 200mM NaCl, 2%CTAB, 1%PVP, pH8.0), add 2 granulated glass spherees (d:1mm), whirlpool 5min fully suspends sample.
Three, the SDS lysis effect detection and the protein removal of different concns
Add 2ml SDS buffer(100mMTirs, 200mM NaCl, 2%SDS, pH8.0) 5min that turns upside down, 4000rpm, centrifugal 10min transfer to supernatant liquor in the new centrifuge tube with the transfer pipet of 1mL.Add the saturated phenol of equal-volume Tris-, put upside down centrifuge tube mixing sample.4000rpm, centrifugal 20min carefully transfer to supernatant in the new centrifuge tube with the transfer pipet of 1mL, note not sucking the protein of white.
If protein is more, repeat above-mentioned steps once.(V (phenol): V (chloroform): V (primary isoamyl alcohol)=25:24:1) mixes to add phenol, chloroform and primary isoamyl alcohol.4000rpm, centrifugal 20min get supernatant, carefully supernatant are transferred in the new centrifuge tube with the pipettor of 1mL.(V (chloroform): V (primary isoamyl alcohol)=24:1) puts upside down mixing, and 4000rpm, centrifugal 20min get supernatant, carefully supernatant is transferred in the new centrifuge tube with the pipettor of 1mL to add chloroform and primary isoamyl alcohol.
Four, the separation of DNA and purification technique
Add 0.6 times of sample volume Virahol, put upside down mixing.Sample is put into the refrigerator precipitation 1h or the precipitation of spending the night.4000rpm, centrifugal 20min outwells supernatant liquor.Sample natural air drying 30min.With 100 μ L TE damping fluid suspended sample, dissolution precipitation, be the genomic dna crude extract of gained.Add 4 μ L RNase A(10mg/mL), 37 ℃, temperature is bathed 15-20min, digestion RNA.Adopt DNA glue to reclaim test kit then, it is carried out purifying according to operation instructions.
Five, the purity testing of DNA
Measure the absorbance of DNA sample respectively with UVmini-1240 model ultraviolet spectrophotometer, calculate OD at 230nm, 260nm and 280nm place 260/ OD 230And OD 260/ OD 280Ratio to determine sample purity.If when containing protein, phenol or other small molecules pollutent in the DNA sample, can influence the accuracy of DNA absorbancy.Can measure the OD of same sample generally speaking 230, OD 260And OD 280Value is calculated the purity that its ratio is weighed its sample again.Pure dna: OD 260/ OD 280≈ 1.8, OD 260/ OD 230＞2; If OD 260/ OD 280＞1.9, then have RNA and pollute; If OD 260/ OD 280＜1.6, then have contaminating impurities such as protein; If OD 260/ OD 230＜2, may there be small molecular weight impurities such as phenols.
Six, DNA electrophoretic analysis
Employing concentration is 1% agarose gel electrophoresis detection, and electrophoretic buffer is 1 * TAE damping fluid; 1 * TAE damping fluid of 100mL contains agarose 1g; Voltage: 110V; About 30 minutes of electrophoresis time; (Ethidium Bromide, EB) final concentration is 1 μ g/mL dyeing 20min, gel imaging system record result with ethidium bromide in dyeing.

Claims (9)

1. the extracting method of active sludge sample DNA among the SBR comprises:
(1) all product of active sludge mixing at first before processing adopts TENP, PBS solution and granulated glass sphere concussion method that sample is carried out pre-treatment; To adopt the freeze-thaw method fragmentation through pretreated mud sample, refrigeration, boiling water boil, and the SDS damping fluid that adds mass percent concentration 2%～10% is again removed protein;
(2) Virahol of 0.6 times of sample volume of adding is put upside down mixing, precipitates; Centrifugal, outwell supernatant liquor; Natural air drying, with TE damping fluid suspended sample, dissolution precipitation promptly gets the genomic dna crude extract; Add RNase A, 35～40 ℃ of temperature are bathed 15-20min digestion RNA, adopt DNA glue to reclaim test kit then, carry out purifying according to operation instructions;
(3) measure the absorption photometric value of above-mentioned purify DNA sample with ultraviolet spectrophotometer at wavelength 230,260,280nm place; Above-mentioned purify DNA sample is carried out agarose gel electrophoresis, behind the ethidium bromide staining, take pictures with the ultraviolet gel imaging system.
2. the extracting method of active sludge sample DNA among a kind of SBR according to claim 1 is characterized in that: TENP liquor capacity in the described step (1) and PBS liquor capacity are 3～5 times of sewage sample; The granulated glass sphere diameter is 1～2mm, the centrifugal 10～30min of 3000～5000rpm.
3. the extracting method of active sludge sample DNA among a kind of SBR according to claim 1, it is characterized in that: refrigeration, boiling water in the described step (1) boil and are specially: place-25～-15 ℃ of refrigerator and cooled to hide 1～2h, boiling water boils 10～20min, repeats once.
4. the extracting method of active sludge sample DNA among a kind of SBR according to claim 1 is characterized in that: after refrigeration, boiling water boil in the described step (1), add 2～4ml Extraction buffer, 1～5 granulated glass sphere, whirlpool 5～10min.
5. the extracting method of active sludge sample DNA among a kind of SBR according to claim 1 is characterized in that: the mass percent concentration of the SDS damping fluid in the described step (1) is 2%.
6. the extracting method of active sludge sample DNA among a kind of SBR according to claim 1 is characterized in that: the precipitation in the described step (2) is specially puts into the refrigerator precipitation 1～2h or the precipitation of spending the night.
7. the extracting method of active sludge sample DNA among a kind of SBR according to claim 1 is characterized in that: the consumption of the TE damping fluid in the described step (2) is 100 μ L.
8. the extracting method of active sludge sample DNA among a kind of SBR according to claim 1 is characterized in that: the concentration of the RNase A in the described step (2) is 10mg/mL, and consumption is 4 μ L.
9. the extracting method of active sludge sample DNA among a kind of SBR according to claim 1 is characterized in that: the sepharose mass percent concentration in the described step (3) is 1%, and voltage is 100～110V, electrophoresis 0.5～1h.
CN2013101513518A 2013-04-26 2013-04-26 Method for extracting activated sludge sample DNA (Deoxyribonucleic Acid) in SBR (Styrene Butadiene Rubber) Pending CN103215254A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102220309A (en) * 2011-04-11 2011-10-19 吉林建筑工程学院 Method for extracting DNA (deoxyribonucleic acid) of active sludge in anaerobic reactor
CN102408161A (en) * 2011-10-14 2012-04-11 南开大学 Biological active early warning marking method for chemical industry wastewater treatment

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102220309A (en) * 2011-04-11 2011-10-19 吉林建筑工程学院 Method for extracting DNA (deoxyribonucleic acid) of active sludge in anaerobic reactor
CN102408161A (en) * 2011-10-14 2012-04-11 南开大学 Biological active early warning marking method for chemical industry wastewater treatment

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
吕娟 等: "厌氧、缺氧、好氧多级交替SBR脱氮除磷试验研究", 《环境污染与防治》, vol. 29, no. 9, 30 September 2007 (2007-09-30), pages 648 - 651 *
廖永红 等: "脱氮活性污泥总DNA提取研究", 《环境科学与技术》, vol. 30, no. 12, 31 December 2007 (2007-12-31), pages 75 - 79 *
沈国 李茵: "废水处理系统中活性污泥总DNA的提取", 《环境工程学报》, vol. 4, no. 6, 30 June 2010 (2010-06-30), pages 1336 - 1340 *
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Application publication date: 20130724